Tricyclic Quaternary Ammonium Salts Derived from Cinchona Alkaloids.

Tricyclic systems with quaternary bridgehead nitrogen atoms are rare but an interesting class of compounds. Chiral quinuclidine derivative salts with fused five and six-membered rings (X-ray) were obtained via modification of Cinchona alkaloids. The ease of ring formation was dependent on its size, while even mild activation sufficed to close the five membered ring. On the other hand the systems with fused benzene and a six-membered ring formed atropisomers separated by a barrier of ca. 15 kcal/mol, whose interconversion was studied by DFT and NMR.

Mechanisms of coupling between DNA recognition specificity and catalysis in EcoRI endonuclease.

Proteins that bind to specific sites on DNA often do so in order to carry out catalysis or specific protein-protein interaction while bound to the recognition site. Functional specificity is enhanced if this second function is coupled to correct DNA site recognition. To analyze the structural and energetic basis of coupling between recognition and catalysis in EcoRI endonuclease, we have studied stereospecific phosphorothioate (PS) or methylphosphonate (PMe) substitutions at the scissile phosphate GpAATTC or at the adjacent phosphate GApATTC in combination with molecular-dynamics simulations of the catalytic center with bound Mg2+. The results show the roles in catalysis of individual phosphoryl oxygens and of DNA distortion and suggest that a "crosstalk ring" in the complex couples recognition to catalysis and couples the two catalytic sites to each other.

Bromodeoxyuridine-labeled oligonucleotides as tools for oligonucleotide uptake studies.

The mechanisms by which various oligonucleotides (ODNs) and their analogs enter cells are not fully understood. A common technique used in studies on cellular uptake of ODNs is their conjugation with fluorochromes. However, fluorescently labeled ODNs may vary from the parent compounds in charge and hydrophilicity, and they may interact differently with some components of cellular membranes. In this report, we present an alternative method based on the immunofluorescent detection of ODNs with incorporated 5-bromo-2'-deoxyuridine (BrdUrd). Localization of BrdUrd-modified ODNs has been achieved using FITC-labeled anti-BrdUrd antibodies. This technique allowed determination of the differences in cellular uptake of phosphodiester (PO) and phosphorothioate (PS) ODNs and their derivatives conjugated with cholesterol and menthol. The immunocytochemical method also has shown that the cellular uptake of some ODNs may be influenced by specific sequences that are responsible for the formation of higher-order structures.